The hanging drop (HD) culture method is widely used for the formation of in vitro 3D aggregation of cells, avoiding the cellular distortion typically seen in the two-dimensional cell culture. The conventional HD technique consists of placing a small drop of medium and cells on a plastic substrate, inverting the substrate, and incubating for the desired length of time. However, the size of the HD is restrained by gravity, and hence only a limited number of cells can be sustained without cumbersome periodic replenishment. Approaches to modified RD method include hollow spheres, 384-well HD culture plates, and microwell structures. In the first two methods, the contents in the HD cannot be exchanged easily. In the microwell system, the substrate must be optimized to support 3D cell growth without cell adhesion